This invention relates to fluorometric systems for the detection of sample substances derived from biological fluid or tissue tagged with fluorochromes and to fluorometers adapted for more accurate measurements of surface mounted fluorescent samples. It is particularly useful in the detection of hormones, enzymes, drugs and other substances.
Most infectious diseases of bacterial or viral nature produce antibodies in the blood serum of the subject. This provides a degree of immunity against future assaults by the identical infectious agent or antigen. One method for detecting the presence of a particular antigen is to add to it a specific antibody which binds to the antigen. If the antibody has been previously tagged with a radioactive element (RIA technique) or a fluorescent dye, which does not interfere with its immunological properties, the coupled comple can be detected by an appropriate detector; and, in the case of the fluorescent additive, can be at best semiquantitatively measured, as is done in cases in the prior art on a microscope slide for visual inspection.
There are many reasons why RIA is not completely satisfactory. For example, in the presence of small quantities of antigen, only few counts per second can be detected. Since the "noise" of the system is proportional to the square root of the signal count, large errors in accuracy are made at low signal levels. Furthermore, radioisotopes have a limited shelf life due to half-life decay, and require special licensing, handling and disposal.
Testing which relies on fluorescence tagging techniques, as heretofore known, has been qualitative, or at best, semiquantitative as an assay. In the area of the largest diagnostic use of fluorometry (i.e., immunofluorescence microscopy in which samples are typically mounted on a microscope stage and illuminated with an exciting wavelength) the fluorescence is observed through the stage with appropriate filters interposed to select the wavelength to be observed. Typically, the resultant observation is recorded by a laboratory technician as a comparative degree of fluorescence, for example 0, +1, +2, +3, or +4 by comparison to known reference concentrations. In some instances, where blood titre or concentration of antibodies is the desired unknown, the technician prepares a number of slides; on each is a different concentration of the test material. Thus, the technician may estimate a +4 reaction in the microscope when the blood serum or the bacteria broth medium was diluted 1:4 in distilled water, or 1:16, or 1:128, etc. It would be of great advantage to medical and clinical authorities if a fluorometer could automatically and quantitatively read titre or concentration quickly and accurately, without the necessity of making serial dilutions.
In liquid scanning fluorometers, a cuvette usually holds a liquid containing the substance to be analyzed and through which excitation light is passed and fluorescence observed in a right angle configuration. It has been found that these systems are unsuitable for the present applications primarily because liquid systems and cuvettes themselves, apart from the sample being investigated all contribute very substantial background fluorescence, so that, unless a very high degree of careful chemical separation is utilized together with extremely well-controlled materials selected to have low fluorescence in the wavelengths of interest, such systems are unsuitable. Attempts to adapt these instruments for surface measurements have not been particularly successful. Such systems are typically inefficient and have not provided for the discrete handling of individual samples. In general, past fluorometers and RIA systems have been unduly sensitive to background and non-sample oriented signals. There is, therefore, a need for a new and improved fluorometric system.